Toll-like receptors (TLRs) form a family of pattern recognition receptors which have a key role in activating the innate immune response. 11 Toll-like receptors have been identified in humans to date. The members of the TLR family are highly conserved, with most mammalian species having between 10 to 15 TLRs. Each TLR recognises specific pathogen-associated molecular signatures. Toll-like receptor 2 (TLR2, CD282, TLR-2) is activated by peptidoglycan, lipoproteins and lipoteichoic acid.
TLRs are transmembrane proteins, in which a single-pass transmembrane α-helix connects the extracellular ligand-binding domain with the intracellular TIR domain. The extracellular domain (ECD) of TLRs is composed of 16-28 consecutive leucine rich repeat (LRR) units forming a bent, horseshoe-like shape. The LRRs stack on top of each other to create a helical or solenoid structure, in which conserved, mostly hydrophobic residues face inward to create a stable core. The conserved regions of consecutive LRRs (denoted by the amino acid sequence LXXLXLXXN, where L stands for leucine or other hydrophobic amino acids, X for any amino acid and N for asparagine) generate a hydrogen-bonded parallel β-sheet at the inner, concave surface of the molecular horseshoe. The variable parts composed of α-helixes and loops, due to their larger size, create the outer, convex surface. The LRR domain of TLRs is protected N- and C-terminal by two small capping modules known as LRR-NT and LRR-CT motifs, which contain cysteine clusters and shield the hydrophobic core formed by the β-sheet from exposure to the solvent.
Extracellular recognition of suitable molecular patterns leads to receptor dimerization which initiates intracellular signalling via cytoplasmic adaptor proteins such as MyD88 culminating in the translocation of the transcription factors (nuclear factor κB or NF-κB and interferon regulatory factor 3 and 7 or IRF3 and 7) into the nucleus and the activation of genes of the immune response. Ligand-induced dimerization of the extracellular domains places the C-termini of the receptors in close mutual proximity causing the transmembrane α-helices to align and the cytoplasmic TIR domains to interact triggering different intracellular signalling cascades. Depending on the ligand, TLR2 interacts with either TLR1 (triacylated lipopeptides) or TLR6 (diacetylated lipopeptides) to create two distinct heterodimers, TLR2/TLR1 and TLR2/TLR6. In both complexes, direct protein-protein interactions involve the central regions near the binding pockets.
In addition to microbial derived components, TLRs are also known to recognize damage-associated molecular patterns (DAMPs). These are host endogenous molecules released and distributed following stress, tissue damage and cellular disease. In cases in which the normal dampening of the immune response and the down-regulation of innate immunity is deregulated, persistent expression of pro-inflammatory cytokines can lead to inflammation and autoimmune diseases such as rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), atherosclerosis and ischemia-reperfusion (I/R) injury. The immune response downstream of TLR2 has implicated TLR2 in many disease conditions. Accordingly, there is significant therapeutic interest in the modulation of the TLR2 signalling pathway and the development of TLR2 antagonists to inhibit cytokine production during inflammation and autoimmunity diseases has become of major interest.
In particular, antibodies have been developed having binding specificity to TLR2. For example, WO 2005/019431 discloses an antibody which has binding specificity to TLR2 designated 11G7. This murine antibody can be derived from hybridoma cell line 11G7 as deposited with the American Type Culture Centre (ATCC) under the designation PTA-5014. The 11G7 monoclonal antibody selectively binds to the extracellular domain of TLR2 and can block the induction of cytokine production by human peripheral blood mononuclear cells (PBMCs) stimulated with an agonist which activates a heterodimer formed between TLR1 and TLR2. The 11G7 antibody does not inhibit cytokine production by PBMCs stimulated with an agonist which induces signalling through a heterodimer formed between TLR6 and TLR2.
WO 2005/028509 discloses the murine T2.5 antibody (also known as OPN-301). This antibody is a murine IgG1 anti-TLR2 antibody which was derived from hybridoma clone T2.5 (HyCult Biotechnology b.v., Cell Sciences, Canton, USA: catalogue number 1054). WO 2005/028509 discloses that T2.5 or OPN-301 specifically inhibits the activation of mammalian TLR2 and cross-reacts with TLR2 from human, pig and monkey, indicating that the antibody is specific for a critical epitope. The T2.5 monoclonal antibody of WO 2005/028509 was raised against the extracellular domain of TLR2, and therefore has binding specificity to an epitope in that area of TLR2. WO 2005/028509 further discloses that the murine TL2.1 anti-TLR2 monoclonal antibody disclosed in WO 01/36488 is not cross-reactive to both human and murine forms of TLR2. Rather, the TL2.1 antibody is shown in WO 2005/028509 as binding only human TLR2 and not murine TLR2.
Several results have shown that TLR2 inhibition with T2.5 effectively reduces myocardial (I/R) injury and preserves cardiac function and geometry in vivo in mice and thus has the potential to be effective when administered to patients with acute myocardial infarction (MI). T2.5 also prevents pro-inflammatory cytokine release in rheumatoid arthritis (RA) tissue synovial explant cultures ex vivo. OPN-305 is a TLR2 specific monoclonal antibody which is a humanized version of T2.5 and which is described in WO2011/003925. OPN-305 was granted orphan status for the prevention of I/R injury associated with organ transplantation and is currently being tested in human trials. It would be desirable to generate further binding members having the same or similar functional properties as the T2.5 (OPN-301) and OPN-305 antibodies.